Hyperubiquitylation of DNA helicase RECQL4 by E3 ligase MITOL prevents mitochondrial entry and potentiates mitophagy.

Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic instability. However, the mechanisms by which these mutations lead to pathology are unclear. Here we report that RECQL4 is ubiquitylated by a mitochondrial E3 ligase, MITOL, at two lysine residues (K1101, K1154) via K6 linkage. This ubiquitylation hampers the interaction of RECQL4 with mitochondrial importer Tom20, thereby restricting its own entry into mitochondria. We show the RECQL4 2K mutant (where both K1101 and K1154 are mutated) has increased entry into mitochondria and demonstrates enhanced mitochondrial DNA (mtDNA) replication. We observed that the three tested RTS patient mutants were unable to enter the mitochondria and showed decreased mtDNA replication. Furthermore, we found that RECQL4 in RTS patient mutants are hyperubiquitylated by MITOL and form insoluble aggregate-like structures on the outer mitochondrial surface. However, depletion of MITOL allows RECQL4 expressed in these RTS mutants to enter mitochondria and rescue mtDNA replication. Finally, we show increased accumulation of hyperubiquitylated RECQL4 outside the mitochondria leads to the cells being potentiated to increased mitophagy. Hence, we conclude regulating the turnover of RECQL4 by MITOL may have a therapeutic effect in patients with RTS.

Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL.

Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and MITOL are used to determine whether PolγA is ubiquitylated. Either anti-ubiquitin or anti-Flag antibody is used to detect the ubiquitylated product. Thus, we provide a detailed, reliable, highly reproducible protocol for detecting ubiquitylation of PolγA by MITOL, both in cells and in vitro. For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021).

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria.

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

'We pledge to improve the health of our entire community': Improving health worker motivation and performance in Bihar, India through teamwork, recognition, and non-financial incentives.

BACKGROUND: Motivation is critical to health worker performance and work quality. In Bihar, India, frontline health workers provide essential health services for the state's poorest citizens. Yet, there is a shortfall of motivated and skilled providers and a lack of coordination between two cadres of frontline health workers and their supervisors. CARE India developed an approach aimed at improving health workers' performance by shifting work culture and strengthening teamwork and motivation. The intervention-"Team-Based Goals and Incentives"-supported health workers to work as teams towards collective goals and rewarded success with public recognition and non-financial incentives. METHODS: Thirty months after initiating the intervention, 885 health workers and 98 supervisors completed an interviewer-administered questionnaire in 38 intervention and 38 control health sub-centers in one district. The questionnaire included measures of social cohesion, teamwork attitudes, self-efficacy, job satisfaction, teamwork behaviors, equitable service delivery, taking initiative, and supervisory support. We conducted bivariate analyses to examine the impact of the intervention on these psychosocial and behavioral outcomes. RESULTS: Results show statistically significant differences across several measures between intervention and control frontline health workers, including improved teamwork (mean = 8.8 vs. 7.3), empowerment (8.5 vs. 7.4), job satisfaction (7.1 vs. 5.99) and equitable service delivery (6.7 vs. 4.99). While fewer significant differences were found for supervisors, they reported improved teamwork (8.4 vs. 5.3), and frontline health workers reported improved fulfillment of supervisory duties by their supervisors (8.9 vs. 7.6). Both frontline health workers and supervisors found public recognition and enhanced teamwork more motivating than the non-financial incentives. CONCLUSIONS: The Team-Based Goals and Incentives model reinforces intrinsic motivation and supports improvements in the teamwork, motivation, and performance of health workers. It offers an approach to practitioners and governments for improving the work environment in a resource-constrained setting and where there are multiple cadres of health workers.

Reference Values for Shear Wave Elastography of Neck and Shoulder Muscles in Healthy Individuals.

PURPOSE: to establish reference values for ultrasound shear-wave elastography for pericranial muscles in healthy individuals (m. trapezius, m. splenius capitis, m. semispinalis capitis, m. sternocleidomastoideus and m. masseter). Also to evaluate day-to-day variations in the shear-wave speeds and evaluate the effect of the pennation of the muscle fibers, ie scanning parallel or perpendicularly to the fibers. MATERIALS AND METHODS: 10 healthy individuals (5 males and 5 females) had their pericranial muscles examined with shear-wave elastography in two orthogonal planes on two different days for their dominant and non-dominant side. Mean shear wave speeds from 5 ROI's in each muscle, for each scan plane for the dominant and non-dominant side for the two days were calculated. The effect of the different parameters - muscle pennation, gender, dominant vs non-dominant side and day was evaluated. RESULTS: The effect of scan plane in relation to muscle pennation was statistically significant (p<0.0001). The mean shear-wave speed when scanning parallel to the muscle fibers was significantly higher than the mean shear-wave speed when scanning perpendicularly to the fibers. The day-to-day variation was statistically significant (p=0.0258), but not clinically relevant. Shear-wave speeds differed significantly between muscles. Mean shear wave speeds (m/s) for the muscles in the parallel plane were: for masseter 2.45 (SD:+/-0.25), semispinal 3.36 (SD:+/-0.75), splenius 3.04 (SD:+/-0.65), sternocleidomastoid 2.75 (SD:+/-0.23), trapezius 3.20 (SD:+/-0.27) and trapezius lateral 3.87 (SD:+/-3.87). CONCLUSION: The shear wave speed variation depended on the direction of scanning. Shear wave elastography may be a method to evaluate muscle stiffness in patients suffering from chronic neck pain.

Systems Biology of Immunomodulation for Post-Stroke Neuroplasticity: Multimodal Implications of Pharmacotherapy and Neurorehabilitation.

AIMS: Recent studies indicate that anti-inflammatory drugs, act as a double-edged sword, not only exacerbating secondary brain injury but also contributing to neurological recovery after stroke. Our aim is to explore whether there is a beneficial role for neuroprotection and functional recovery using anti-inflammatory drug along with neurorehabilitation therapy using transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS), so as to improve functional recovery after ischemic stroke. METHODS: We develop a computational systems biology approach from preclinical data, using ordinary differential equations, to study the behavior of both phenotypes of microglia, such as M1 type (pro-inflammatory) vis-à-vis M2 type (anti-inflammatory) under anti-inflammatory drug action (minocycline). We explore whether pharmacological treatment along with cerebral stimulation using tDCS and rTMS is beneficial or not. We utilize the systems pathway analysis of minocycline in nuclear factor kappa beta (NF-κB) signaling and neurorehabilitation therapy using tDCS and rTMS that act through brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) signaling pathways. RESULTS: We demarcate the role of neuroinflammation and immunomodulation in post-stroke recovery, under minocycline activated-microglia and neuroprotection together with improved neurogenesis, synaptogenesis, and functional recovery under the action of rTMS or tDCS. We elucidate the feasibility of utilizing rTMS/tDCS to increase neuroprotection across the reperfusion stage during minocycline administration. We delineate that the signaling pathways of minocycline by modulation of inflammatory genes in NF-κB and proteins activated by tDCS and rTMS through BDNF, TrkB, and calmodulin kinase (CaMK) signaling. Utilizing systems biology approach, we show that the activation pathways for pharmacotherapy (minocycline) and neurorehabilitation (rTMS applied to ipsilesional cortex and tDCS) results into increased neuronal and synaptic activity that commonly occur through activation of N-methyl-d-aspartate receptors. We construe that considerable additive neuroprotection effect would be obtained and delayed reperfusion injury can be remedied, if one uses multimodal intervention of minocycline together with tDCS and rTMS. CONCLUSION: Additive beneficial effect is, thus, noticed for pharmacotherapy along with neurorehabilitation therapy, by maneuvering the dynamics of immunomodulation using anti-inflammatory drug and cerebral stimulation for augmenting the functional recovery after stroke, which may engender clinical applicability for enhancing plasticity, rehabilitation, and neurorestoration.

The incidence and visual acuity outcomes of children identified with ametropic amblyopia by vision screening.

PURPOSE: To determine the incidence of ametropic amblyopia within a vision screening program's population and report the visual acuity outcomes of children identified with the condition. METHOD: The medical records of children who underwent vision screening as their first assessment at 4-5 years of age between September 1, 2005 and August 31, 2006, were retrospectively reviewed. Children referred with ≤0.30 logMAR in each eye with at least 1 year of follow-up had their hospital notes reviewed and data on final visual acuity, refractive error, and follow-up period collected. RESULTS: A total of 33 children identified as having ametropic amblyopia with a follow-up of at least 1 year. The incidence of ametropic amblyopia was 2%-3.2%, depending on the definition used. The mean visual acuity achieved after treatment was 0.12 logMAR, which is significantly less than the age-appropriate mean of 0.00 logMAR (P < 0.01). CONCLUSIONS: Ametropic amblyopia responds to treatment, but most children demonstrate persistent reduced visual acuity at age 7 years. The incidence of ametropic amblyopia within a routine vision screening population shows that significant numbers fail to self-present.

Drug delivery using platelet cancer cell interaction.

PURPOSE: To develop an efficient biocompatible and targeted drug delivery system in which platelets, an essential blood component having a natural affinity for cancer cells, are used as carrier of anticancer drug as delivery of drug to the targeted site is crucial for cancer treatment. METHODS: Doxorubicin hydrochloride, a potent anti cancer drug, was delivered in lung adenocarcinoma cell line (A549) using platelet as a delivery agent. This delivery mode was also tested in Ehrlich ascites carcinoma (EAC) bearing mice in presence and absence of platelets. RESULTS: The results show that platelets can uptake the drug and release the same upon activation. The efficiency of drug loaded platelets in inducing cytotoxicity was significantly higher in both in vitro and in vivo model, as compared to the free drug. CONCLUSIONS: The proposed drug delivery strategy may lead to clinical improvement in the management of cancer treatment as lower drug concentration can be used in a targeted mode. Additionally the method can be personalized as patient's own platelet can be used for deliver various drugs.

A high molecular weight protein extract of Mastobranchus indicus (Mi-64) having antiarthritic activity in experimental animals.

Mi-64, a high molecular weight protein (130 kDa), obtained from the tissue homogenate of marine polychaete (Mastobranchus indicus) collected from the Indian Sunderban has antiarthritic activity in experimental animals. The FCA-induced arthritis model was developed in Wistar albino rats to evaluate the antiarthritic effects of Mi-64. After FCA induction, the rats were treated with Mi-64 (0.25 and 0.5 mg kg(-1) body weight) for 10 days. We have determined the paw/ankle swellings, urinary hydroxyproline and glucosamine, serum acid and alkaline phosphatases to assess the antiarthritic activity. The levels of interleukin-1 beta (IL-1ß), IL-6, cytokine-induced neutrophil chemoattractant-1 (CINC-1), tumor necrosis factor-alpha (TNF-α), and IL-10 were measured by ELISA. Results showed that Mi-64 significantly reduced paw/ankle swellings and restored the urinary hydroxyproline/glucosamine and serum phosphatases. Mi-64 significantly inhibited the overproduction of IL-1ß, IL-6, CINC-1, and TNF-α and augmented IL-10 production. The data suggest that Mi-64 produced significant antiarthritic effects that may be mediated by balancing the pro- and anti-inflammatory cytokines.

Hepatoprotective activity of the edible snail (Bellamia bengalensis) flesh extract in carbon tetrachloride induced hepatotoxicity in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In the folk-traditional medicine, snails were used to purify blood, boost immune system, prevent conjunctivitis and to treat liver problems. OBJECTIVES: To evaluate the hepatoprotective activity of the edible snail (Bellamia bengalensis) flesh extract in male Wistar rats treated with carbon tetrachloride as an hepatotoxicant. MATERIALS AND METHODS: Live adult Bellamia bengalensis was collected commercially from the Kolkata market. Aqueous flesh extract (BBE) was prepared in 0.9% saline and expressed in terms of wet weight basis. The aqueous flesh extract was administered orally (1, 2 g kg(-1)day(-1)) to male rats for 12 days. Liv52 was used as positive control. 24h after administration of extract, the rats were given a single oral dose of CCl(4) (1.25 ml kg(-1)), except vehicle control rats. After 24h of CCl(4) administration, all the animals were sacrificed to collect the blood and liver tissue. RESULTS: BBE (1 and 2 g kg(-1)day(-1), p.o.×12 days) significantly prevented CCl(4) induced elevation of SGOT, SGPT, γGT, ACP, ALP, bilirubin, LDH and CCl(4) induced decrease in total protein, triglyceride level in male Wistar rats. BBE treated rat liver anti-oxidant parameters (LPO, SOD, GSH, CAT, GPx) were significantly antagonized for the pro-oxidant effect of CCl(4). Histopathological studies also supported the protective effect of BBE. CONCLUSION: This study validated the folk and traditional use of snail in liver disorder through CCl(4)-induced rat experimental model.